Varying Selection Pressure for a Na+ Sensing Site in Epithelial Na+ Channel Subunits Reflect Divergent Roles in Na+ Homeostasis.

The epithelial Na+ channel (ENaC) emerged early in vertebrates and has played a role in Na+ and fluid homeostasis throughout vertebrate evolution. We previously showed that proteolytic activation of the channel evolved at the water-to-land transition of vertebrates. Sensitivity to extracellular Na+, known as Na+ self-inhibition, reduces ENaC function when Na+ concentrations are high and is a distinctive feature of the channel. A fourth ENaC subunit, δ, emerged in jawed fishes from an α subunit gene duplication. Here, we analyzed 849 α and δ subunit sequences and found that a key Asp in a postulated Na+ binding site was nearly always present in the α subunit, but frequently lost in the δ subunit (e.g. human). Analysis of site evolution and codon substitution rates provide evidence that the ancestral α subunit had the site and that purifying selection for the site relaxed in the δ subunit after its divergence from the α subunit, coinciding with a loss of δ subunit expression in renal tissues. We also show that the proposed Na+ binding site in the α subunit is a bona fide site by conferring novel function to channels comprising human δ subunits. Together, our findings provide evidence that ENaC Na+ self-inhibition improves fitness through its role in Na+ homeostasis in vertebrates.

Heterogenous thinning of peripapillary tissues occurs early during high myopia development in juvenile tree shrews.

Myopia is an independent risk factor for glaucoma, but the link between both conditions remains unknown. Both conditions induce connective tissue remodeling at the optic nerve head (ONH), including the peripapillary tissues. The purpose of this study was to investigate the thickness changes of the peripapillary tissues during experimental high myopia development in juvenile tree shrews. Six juvenile tree shrews experienced binocular normal vision, while nine received monocular -10D lens treatment starting at 24 days of visual experience (DVE) to induce high myopia in one eye and the other eye served as control. Daily refractive and biometric measurements and weekly optical coherence tomography scans of the ONH were obtained for five weeks. Peripapillary sclera (Scl), choroid-retinal pigment epithelium complex (Ch-RPE), retinal nerve fiber layer (RNFL), and remaining retinal layers (RRL) were auto-segmented using a deep learning algorithm after nonlinear distortion correction. Peripapillary thickness values were quantified from 3D reconstructed segmentations. All lens-treated eyes developed high myopia (-9.8 ± 1.5 D), significantly different (P < 0.001) from normal (0.69 ± 0.45 D) and control eyes (0.76 ± 1.44 D). Myopic eyes showed significant thinning of all peripapillary tissues compared to both, normal and control eyes (P < 0.001). At the experimental end point, the relative thinning from baseline was heterogeneous across tissues and significantly more pronounced in the Scl (-8.95 ± 3.1%) and Ch-RPE (-16.8 ± 5.8%) when compared to the RNFL (-5.5 ± 1.6%) and RRL (-6.7 ± 1.8%). Furthermore, while axial length increased significantly throughout the five weeks of lens wear, significant peripapillary tissue thinning occurred only during the first week of the experiment (until a refraction of -2.5 ± 1.9 D was reached) and ceased thereafter. A sectorial analysis revealed no clear pattern. In conclusion, our data show that in juvenile tree shrews, experimental high myopia induces significant and heterogeneous thinning of the peripapillary tissues, where the retina seems to be protected from profound thickness changes as seen in Ch-RPE and Scl. Peripapillary tissue thinning occurs early during high myopia development despite continued progression of axial elongation. The observed heterogeneous thinning may contribute to the increased risk for pathological optic nerve head remodeling and glaucoma later in life.

A novel tree shrew model of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective therapy. Animal models effectively reproducing IPF disease features are needed to study the underlying molecular mechanisms. Tree shrews are genetically, anatomically, and metabolically closer to humans than rodents or dogs; therefore, the tree shrew model presents a unique opportunity for translational research in lung fibrosis. Here we demonstrate that tree shrews have in vivo and in vitro fibrotic responses induced by bleomycin and pro-fibrotic mediators. Bleomycin exposure induced lung fibrosis evidenced by histological and biochemical fibrotic changes. In primary tree shrew lung fibroblasts, transforming growth factor beta-1 (TGF-ß1) induced myofibroblast differentiation, increased extracellular matrix (ECM) protein production, and focal adhesion kinase (FAK) activation. Tree shrew lung fibroblasts showed enhanced migration and increased matrix invasion in response to platelet derived growth factor BB (PDGF-BB). Inhibition of FAK significantly attenuated pro-fibrotic responses in lung fibroblasts. The data demonstrate that tree shrews have in vivo and in vitro fibrotic responses similar to that observed in IPF. The data, for the first time, support that the tree shrew model of lung fibrosis is a new and promising experimental animal model for studying the pathophysiology and therapeutics of lung fibrosis.

Histologic validation of optical coherence tomography-based three-dimensional morphometric measurements of the human optic nerve head: Methodology and preliminary results.

PURPOSE: To compare the three-dimensional (3D) morphology of the deep load-bearing structures of the human optic nerve head (ONH) as revealed in vivo by spectral domain optical coherence tomography (SDOCT) with ex vivo quantitative 3D histology. METHODS: SDOCT imaging of the ONH was performed in six eyes from three brain-dead organ donors on life-support equipment awaiting organ procurement (in vivo conditions). Following organ procurement (ex vivo conditions), the eyes were enucleated and underwent a pars plana vitrectomy followed by pressurization to physiologic IOP and immersion fixation. Ex vivo ONH morphology was obtained from high-fidelity episcopic fluorescent 3D reconstruction. Morphologic parameters of the observed ONH canal geometry and peripapillary choroid, as well as the shape, visibility and depth of the lamina cribrosa were compared between ex vivo and in vivo measurements using custom software to align, scale, and manually delineate the different regions of the ONH. RESULTS: There was significant correspondence between in vivo and ex vivo measurements of the depth and shape of the lamina cribrosa, along with the size and shape of Bruch's membrane opening (BMO) and anterior scleral canal opening (ASCO). Weaker correspondence was observed for choroidal thickness; as expected, a thinner choroid was seen ex vivo due to loss of blood volume upon enucleation (-79.9%, p < 0.001). In addition, the lamina was shallower (-32.3%, p = 0.0019) and BMO was smaller ex vivo (-3.38%, p = 0.026), suggesting post mortem shrinkage of the fixed tissue. On average, while highly variable, only 31% of the anterior laminar surface was visible in vivo with SDOCT (p < 0.001). CONCLUSIONS: Morphologic parameters by SDOCT imaging of the deep ONH showed promising correspondence to histology metrics. Small but significant shrinkage artifact, along with large effects of exsanguination of the choroid, was seen in the ex vivo reconstructions of fixed tissues that may impact the quantification of ex vivo histoarchitecture, and this should be considered when developing models and biomarkers based on ex vivo imaging of fixed tissue. Lack of visibly of most of the lamina surface in SDOCT images is an important limitation to metrics and biomarkers based on in vivo images of the ONH deep tissues.

Matching the LenStar optical biometer to A-Scan ultrasonography for use in small animal eyes with application to tree shrews.

We describe an analysis strategy to obtain ultrasonography-matched axial dimensions of small animal eyes using the LenStar biometer. The LenStar optical low-coherence reflectometer is an attractive device for animal research due to its high precision, non-invasiveness, and the ability to measure the axial dimensions of cornea, anterior chamber, lens, vitreous chamber, and axial length. However, this optical biometer was designed for clinical applications in human eyes and its internal analysis provides inaccurate values when used on small eyes due to species-dependent differences in refractive indices and relative axial dimensions. The LenStar uses a near infrared light source to measure optical path lengths (OPLs) that are converted by the LenStar's EyeSuite software into geometrical lengths (GLs) based on the refractive indices and axial dimensions of the human eye. We present a strategy that extracts the OPLs, determines refractive indices specific for the small animal eye of interest and then calculates corrected GLs. The refractive indices are obtained by matching the LenStar values to ultrasonography values in the same eyes. As compared to ultrasounography, we found that the internal calculations of the LenStar underestimate the axial dimensions of all ocular compartments of the tree shrew eye: anterior segment depth by 6.17±4.50%, lens thickness by 1.37±3.06%, vitreous chamber depth by 29.23±2.35%, and axial length by 10.62±1.75%. Using tree shrew-specific refractive indices, the axial dimensions closely matched those measured by ultrasonography for each compartment. Our analysis strategy can be easily translated to other species by obtaining a similar paired data set using ultrasonography and LenStar, and applying our step by step procedures.

Longitudinal Changes of Bruch's Membrane Opening, Anterior Scleral Canal Opening, and Border Tissue in Experimental Juvenile High Myopia.

Purpose: To investigate the relative positional changes between the Bruch's membrane opening (BMO) and the anterior scleral canal opening (ASCO), and border tissue configuration changes during experimental high myopia development in juvenile tree shrews. Methods: Juvenile tree shrews were assigned randomly to two groups: binocular normal vision (n = 9) and monocular -10 D lens treatment starting at 24 days of visual experience to induce high myopia in one eye while the other eye served as control (n = 12). Refractive and biometric measurements were obtained daily, and 48 radial optical coherence tomography B-scans through the center of the optic nerve head were obtained weekly for 6 weeks. ASCO and BMO were segmented manually after nonlinear distortion correction. Results: Lens-treated eyes developed high degree of axial myopia (-9.76 ± 1.19 D), significantly different (P < 0.001) from normal (0.34 ± 0.97 D) and control eyes (0.39 ± 0.88 D). ASCO-BMO centroid offset gradually increased and became significantly larger in the experimental high myopia group compared with normal and control eyes (P < 0.0001) with an inferonasal directional preference. The border tissue showed a significantly higher tendency of change from internally to externally oblique configuration in the experimental high myopic eyes in four sectors: nasal, inferonasal, inferior, and inferotemporal (P < 0.005). Conclusions: During experimental high myopia development, progressive relative deformations of ASCO and BMO occur simultaneously with changes in border tissue configuration from internally to externally oblique in sectors that are close to the posterior pole (nasal in tree shrews). These asymmetric changes may contribute to pathologic optic nerve head remodeling and an increased risk of glaucoma later in life.

Nonlinear distortion correction for posterior eye segment optical coherence tomography with application to tree shrews.

We propose an empirical distortion correction approach for optical coherence tomography (OCT) devices that use a fan-scanning pattern to image the posterior eye segment. Two types of reference markers were used to empirically estimate the distortion correction approach in tree shrew eyes: retinal curvature from MRI images and implanted glass beads of known diameter. Performance was tested by correcting distorted images of the optic nerve head. In small animal eyes, our purposed method effectively reduced nonlinear distortions compared to a linear scaling method. No commercial posterior segment OCT provides anatomically correct images, which may bias the 3D interpretation of these scans. Our method can effectively reduce such bias.

Effect of Scleral Crosslinking Using Multiple Doses of Genipin on Experimental Progressive Myopia in Tree Shrews.

Purpose: To evaluate the effect of scleral crosslinking (SXL) on slowing experimental progressive myopia in tree shrew eyes using sub-Tenon's injections of genipin (GEN) at different concentrations and number of injections. Methods: Three or five sub-Tenon's injections of GEN at 0 mM (sham), 10 mM, or 20 mM were performed in one eye every other day starting at 18 days of visual experience. Form deprivation (FD) myopia was induced in the injected eye between 24 and 35 days of visual experience; the fellow eye served as control. Tree shrews were randomly assigned to five experimental groups: FD (n = 8); FD + 5 × sham injections (n = 6); FD + 3 × GEN injections at 10 mM (n = 6) and 20 mM (n = 6); and FD + 5 × GEN injections at 20 mM (n = 6). Refractive state and ocular dimensions were measured daily. Results: Compared with the FD group, the sham-injected group showed a transient effect on slowing vitreous chamber elongation. With increasing GEN dose, SXL had an increasing treatment effect on slowing vitreous chamber elongation and myopia progression. In addition, SXL led to a dose-dependent shortening of the aqueous chamber depth and corneal thickening. Lens thickening was observed in the group with the highest concentration. Conclusions: We have shown that SXL using GEN can slow axial elongation and myopia progression in tree shrews. The extent of this treatment effect was dose dependent. Several unexpected effects were observed (corneal thickening, decrease of the anterior chamber depth, and lens thickening), which require further optimization of the GEN delivery approach before clinical consideration. Translational Relevance: The results of this preclinical study suggest that scleral crosslinking using genipin can slow myopia progression.

Technical advance: The use of tree shrews as a model of pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease with a high morbidity and mortality. Some of the mechanisms of fibrosis development have been described using rodent models; however, the relevance of findings in these animal models is difficult to assess. New innovative models are needed that closely mimic IPF disease pathology. METHODS: To overcome this unmet need of investigating IPF with a relevant model, we utilized tree shrews, which are genetically, anatomically, and metabolically similar to primates and humans. Using human antibodies and primers, we investigated the role of macrophage phenotypic switching in normal and IPF subjects and bleomycin-injured tree shrews. RESULTS: Bronchoalveolar lavage (BAL) cells from tree shrews expressed human markers, and there was recruitment of monocyte-derived macrophages (MDMs) to the lung in IPF subjects and bleomycin-injured tree shrews. MDMs were polarized to a profibrotic phenotype in IPF and in bleomycin-injured tree shrews. Resident alveolar macrophages (RAMs) expressed proinflammatory markers regardless of bleomycin exposure. Tree shrews developed bleomycin-induced pulmonary fibrosis with architectural distortion in parenchyma and widespread collagen deposition. CONCLUSION: The profibrotic polarization of macrophages has been demonstrated to be present in IPF subjects and in fibrotic mice. Although the lung macrophages have long been considered to be homogeneous, recent evidence indicates that these cells are heterogeneous during multiple chronic lung diseases. Here, we show new data that indicate a critical and essential role for macrophage-fibroblast crosstalk promoting fibroblast differentiation and collagen production. in the development and progression of fibrosis. The current data strongly suggest development of therapeutics that attenuate of the profibrotic activation of MDMs may mitigate macrophage-fibroblast interaction. These observations demonstrate that tree shrews are an ideal animal model to investigate the pathogenesis of IPF as they are genetically, anatomically, and metabolically closer to humans than the more commonly used rodent models.

Multi-Scale Modeling of Vision-Guided Remodeling and Age-Dependent Growth of the Tree Shrew Sclera During Eye Development and Lens-Induced Myopia.

The sclera uses unknown mechanisms to match the eye's axial length to its optics during development, producing eyes with good focus (emmetropia). A myopic eye is too long for its own optics. We propose a multi-scale computational model to simulate eye development based on the assumption that scleral growth is controlled by genetic factors while scleral remodeling is driven by genetic factors and the eye's refractive error. We define growth as a mechanism that changes the tissue volume and mass while remodeling involves internal micro-deformations that are volume-preserving at the macroscale. The model was fitted against longitudinal refractive measurements in tree shrews of different ages and exposed to three different visual conditions: (i) normal development; (ii) negative lens wear to induce myopia; and (iii) recovery from myopia by removing the negative lens. The model was able to replicate the age- and vision-dependent response of the tree shrew experiments. Scleral growth ceased at younger age than scleral remodeling. The remodeling rate decreased as the eye emmetropized but increased at any age when a negative lens was put on. The predictive power of the model was investigated by calculating the susceptibility to scleral remodeling and the response to form deprivation myopia in tree shrews. Both predictions were in good agreement with experimental data that were not used to fit the model. We propose the first model that distinguishes scleral growth from remodeling. The good agreement of our results with experimental data supports the notion that scleral growth and scleral remodeling are two independently controlled mechanisms during eye development.